1. Field of the Invention
The present invention relates generally to the fields of cancer therapy. More particularly, the invention concerns the use of small antisense oligodeoxynucleotides for antitumor therapy.
2. Description of Related Art
The bcl-2 gene has been associated with a wide variety of diseases such as hematologic malignancies and includes both leukemias and lymphomas and more specifically includes follicular and nonfollicular lymphomas, chronic lymphocytic leukemia, and plasma cell dyscrasias (Campos et al., 1994); solid tumors like those associated with breast, prostate and colon cancer; and immune disorders. One particular Bcl-2-related disease is Follicular non-Hodgkin Lymphoma (FL). Follicular lymphoma is the most common lymphoid malignancy in Europe and the United States. Typically it is an indolent, low grade disease consisting of an accumulation of small, resting B cells. Although the initial response to chemotherapy is good, relapses are inevitable and the disease transforms to a more aggressive histological type and develops drug resistance (Aisenberg, 1995; Johnson et al., 1995).
In over 90% of follicular lymphoma patients, a t(14;18) translocation is found, which results in the juxtaposition of the bcl-2 gene from chromosome 18q21 with the immunoglobulin heavy chain gene locus on chromosome 14q323 (Tsujimoto et al., 1985; Graninger et al., 1987). As a consequence, the bcl-2 gene comes under the influence of an immunoglobulin heavy chain enhancer, resulting in the overexpression of the Bcl-2 protein (Bakhshi et al., 1985; Tsujimoto et al., 1987). The tumorigenic potential of Bcl-2 is related to its capacity of interfering with physiological cell-death responses, thereby enhancing the longevity of the cell (Nuñez et al., 1990). The Bcl-2 protein blocks apoptotic stimuli such as growth factor deprivation, radiation, heat-shock, virus, and most DNA damaging agents for example, most chemotherapeutic agents (Reed, 1995; Hockenbery et al., 1990). In bcl-2-Ig-transgenic mice, a polyclonal follicular lymphoproliferation consisting of an expansion of mature B lymphocytes is initially observed (McDonnell et al., 1989). Subsequently, monoclonal high grade large immunoblastic type lymphomas develop and about 50% of them present rearrangement of C-MYC. This suggests that a second genetic alteration is necessary for the development and progression of malignant lymphoma (McDonnell and Korsmeyer, 1991).
An expanding family of Bcl-2-related proteins have been identified and include Bax, Bcl-XL, Bcl-XS, Bad, Bak, Mcl-1, A-1, and several open reading frames of DNA viruses (Oltvai et al., 1993; Boise et al., 1993; Yang et al., 1995; Chittenden et al., 1995; Kiefer et al., 1995; Kozopas et al., 1993; Lin et al., 1993; Pearson et al., 1987; Neilan et al., 1993). Membership in the Bcl-2 family of proteins is principally defined by homology within the BH1 and BH2 domains, which help regulate dimerization between the members (Sato et al., 1994). Bax, which shares 21% amino-acid identity with Bcl-2, can bind to Bcl-2 protein and neutralize its ability to block cell death. Thus, the ratio of Bcl-2 to Bax is thought to determine the cell's susceptibility to death following an apoptotic stimulus (Oltvai et al., 1993; Yin et al., 1994). U.S. Pat. No. 5,837,838 to Reed et al., 1998c, provides methods for identifying agents that can modulate the binding of a Bax-inhibitor protein to a member of the Bcl-2 family of proteins.
Phosphodiester antisense oligodeoxynucleotides complementary to specific sequences of the translation-initiation site of Bcl-2 mRNA are able to inhibit the production of the Bcl-2 protein and thereby inhibit the growth of t(14;18) translocation bearing cells (Kitada et al., 1993). However, therapeutic use of phosphodiester oligonucleotides is hampered by their low cellular uptake and their rapid degradation by nucleases and other serum or cellular components. Phosphorothioate oligonucleotides, which are resistant to nuclease degradation, were found to inhibit follicular lymphoma cell growth at concentrations 10 times lower than phosphodiester oligonucleotides (Reed et al., 1990a; Reed et al., 1990b; Cotter et al., 1994). However, this approach suffers from low cellular uptake of the oligonucleotides. For example, Reed et al., (1990a) and Reed et al., (1998a), had to use concentrations of greater than 25 μM of phosphorothioates to achieve 50% growth inhibitions of human leukemic cell-lines Su-Dhl-4, RS11846, 679 and JURKAT and in human PBL (peripheral blood lymphocytes).
Incorporation of oligonucleotides into liposomes has increased their uptake into leukemic cells (Akhtar et al., 1991; Tari et al., 1994). The use of cationic lipids by Reed et al., to deliver phosphorothioate antisense oligonucleotides allowed them to reduce the concentration of oligonucleotides to 0.075 to 0.3 μM and still induce growth inhibition in Su-Dhl-4 cells.
In a related invention, disclosed in U.S. patent application Ser. No. 09/112,869, filed Jul. 9, 1998, the present inventors describe various liposomal compositions of antisense oligonucleotides and methods of making these compositions. The application also describes the use of these liposomal compositions to deliver antisense oligonucleotides to tumor cells and methods for inhibiting the growth of tumor cells.
U.S. Pat. No. 5,734,033 (Reed et. al., 1998a), reports the use of antisense oligonucleotide sequences derived from regions of the translation-initiation site of the bcl-2 gene which are 10 bases or greater in length for the inhibition of growth of leukemic cells and human PBL cells. However, there are no examples demonstrating the synthesis and use of antisense oligonucleotides shorter than 15-mers. Also, both the phosphorothioate and the phosphodiester antisense oligonucleotides were required at concentrations greater than 25 μM for the inhibition of about 50% of cell growth in the human leukemic cell-lines RS11846, 679 and JURKAT and in human PBL (peripheral blood lymphocytes).
Related U.S. Pat. No. 5,831,066 to Reed (1998b), proposes that antisense oligomers of from 2 to 200 nucleotides in length will bind to a human bcl-2 mRNA at the translation initiation site and reduce bcl-2 expression in tumor cells. However, again, there is no disclosure reciting the synthesis and successful use of antisense oligonucleotides shorter than 15 mers.
There is, therefore, a great need for better compositions for the treatment of Bcl-2 associated diseases such as hematologic malignancies, both leukemias and lymphomas, including follicular and nonfollicular lymphomas, chronic lymphocytic leukemia, and plasma cell dyscrasias; solid tumors like those associated with breast, prostate and colon cancer; and immune disorders.